Diagnostic screens for type 1 diabetes (IDDM)

ABSTRACT

The present invention features methods for diagnosing insulin-dependent diabetes mellitus (IDDM) in a test subject, for example a subject at risk of developing IDDM, which involve detecting hyper-responsive Ca 2+  mobilization in cells obtained from the test subject. Hyper-responsive Ca 2+  mobilization can be detected in cells contacted with a stimulatory agent, preferably in cells contacted with a potentiating agent and a stimulatory agent. Methods of the present invention include comparing Ca 2+  mobilization in cells from the test subject to Ca 2+  mobilization in cells from a control subject. The present invention further features kits for use in the detection of IDDM.

This application claims the benefit of Provisional No. 60/097,518 filedAug. 20, 1998.

BACKGROUND OF THE INVENTION

Approximately 16 million people (roughly 6% of the population) in theUnited States suffer from diabetes. Diabetes is the seventh leadingcause of death (sixth leading cause of death by disease) in the UnitedStates claiming approximately 200,000 lives each year. Moreover,diabetes is one of the most costly health problems in America, runningupwards of $92 billion in health care costs annually. Life-threateningcomplications associated with diabetes include cardiovascular diseaseand stroke, high blood pressure, blindness, kidney disease, nervedisease and amputation. Of the 16 million diabetics in the UnitedStates, approximately 5-10% suffer from IDDM (Insulin-Dependent DiabetesMellitus) otherwise known as Type 1 diabetes. At least 30,000 new casesof IDDM are diagnosed each year. Persons with IDDM fail to produceinsulin and, accordingly, are required to take daily insulin injectionsin order to stay alive. Many people are unaware that they have diabetesuntil they develop one or more of its life-threatening complications.Accordingly, much biomedical research has focussed on the cause anddevelopment of diabetes with the hope that having a better understandingof the disease will ultimately aid in earlier detection and/or bettertherapeutic treatments.

With regards to IDDM or Type 1 diabetes, three major theories have beenadvanced to account for the pathogenesis of the disease. The first isthat IDDM is an inherited, or genetic disease. The second, that IDDMresults from autoimmunity. The third theory states that IDDM is broughtabout by an environmental insult, presumably viral (Cotran (1989)Robbins Pathologic Basis of Disease 994-1005; Foster (1991) Harrison'sPrinciples of Internal Medicine 1739-1759). Most agree however, that itis a combination of elements of all three theories that eventuates inIDDM, rather than each of the three acting independently in differentindividuals.

There is much evidence to support the theory that IDDM is an inheriteddisease. IDDM tends to aggregate in families, meaning that if oneindividual in a given family has the disease, each other member of thefamily has a greater chance of developing it. Certain HLA types, notablythose of the D region of chromosome 6, carry an increased risk of IDDM(Cox et al. (1994) Diabetologia 37:500-503). Despite the presence ofthis genetic evidence, the facts remain that IDDM has a low prevalenceof direct vertical transmission, and that the concordance rate of IDDMin monozygotic twins is only 20% (Cotran supra; Foster supra). Thisindicates that something more complex than simple Mendelian genetics isoperating to cause the disease.

Several features of the pathogenesis of IDDM, resemble those ofautoimmune diseases. Notably, patients newly diagnosed with IDDM haveinfiltration of the islets with activated T lymphocytes and antibodiesdirected against islet cell antigens, which are also present in theserum of non-diabetic siblings destined to develop the disease (Fostersupra).

The third theory of the development of IDDM holds that diabetes resultsfrom environmental insult. Certain toxins can result in destruction ofthe pancreas, but the more likely offending agent is a virus. Infectionwith coxsackie B virus, congenital rubella, measles, mumps,cytomegalovirus, hepatitis, and infectious mononucleosis all carryincreased risk of subsequent development of IDDM (Cotran supra; Fostersupra). Pancreatic infection with one of these viral agents could bringabout β-cell destruction via direct inflammatory disruption, or byinduction of an immune response (Foster supra).

More likely than one or the other of these theories explaining IDDM in agiven individual is the combination of the three hypothesized causesparticipating in a sequence of events which results in the destructionof the β-cell, and overt IDDM (Foster supra). A viral infection in agenetically predisposed individual could bring about an inappropriatelylarge inflammatory response in the pancreas. Local inflammation canbring about the increased expression of novel MHC molecules on thesurface of islet cells. In particular, the cytokines TNF-α and IL-1β,important players in the inflammatory response, have been shown toincrease MHC expression on pancreatic β-cells (Campbell and Harrison(1989) J. Cell Biochem. 40:57-66; Han et al. (1996) J. Autoimmunol.9:331-339; Picarella et al. (1993) J. Immunol. 150:4136-4150; Ohashi etal. (1993) J. Immunol. 150:5185-5194). Novel MHC expression could bringabout the eventual antibody formation and autoimmune destruction of theβ-cells, with IDDM as the result.

IDDM results from destruction of the insulin-producing β-cells of thepancreatic islets. Without insulin, glucose is not effectively taken upinto such metabolically active tissues as muscle, liver or adiposetissue. The results is hyperglycemia. Fasting blood glucose levelsgreater than 7.8 mM, or non-fasting levels greater than 11 mM result inthe diagnosis of diabetes. Although levels of blood glucose are veryhigh in uncontrolled diabetics, the body senses a “starved” state, andbegins to release free fatty acids from adipose tissue. The blood levelsof free fatty acids in the early stages of ketoacidosis can be in excessof 2 mM. Fatty acids are used as fuel by the liver and by muscle tissuebecause they can enter the cell freely, whereas glucose can no longerenter due to lack of insulin. The insulin deficiency, together withelevated free fatty acids stimulates gluconeogenesis, furtherexacerbating the hyperglycemia. The abundance of fatty acid oxidationoccurring in the liver leads to an excess production of acetyle CoA. Theexcess acetyl CoA is converted into ketone bodies. Elevation andunderutilization of ketone bodies and fatty acids produces a metabolicacidosis, termed diabetic ketoacidosis, which can progress to coma anddeath if not treated with insulin. The hyperglycemia present in IDDM isthought to contribute to the major pathologies associated with thedisease, such as those found in the peripheral nerves, retina, kidney,and vasculature.

Although IDDM patients may have grossly elevated serum levels of freefatty acids, much less is known about how this may contribute todiabetic pathology than is known about hyperglycemia-related pathology.Even in non-ketotic states, IDDM patients have dyslipidemia, or elevatedlevels of fatty acid in the serum (Azad et al. (1994) Arch. Dis.Childhood 71:108-113). Following insulin-induced hypoglycemia,stimulation of diabetics with epinephrine results in increased freefatty acids greater than in controls subjected to the same maneuver(Bolinder et al. (1996) Diabetologia 39:845-853; Cohen et al. (1996) Am.J. Physiol. 271 :E284-293). Certain fatty acids have effects on variouscells ranging from modulation of intracellular Ca²⁺ homeostatis (Deeneyet al. (1992) J. Biol. Chem. 267:19840-19845, to activation of thenuclear transcription factor NF-kB, and modulation of gene expression(Prentki et al. (1997) Diabetologia 40 Suppl 2:S32-41; Prentki andCorkey (1996) Diabetes 45:273-283). Elevated extracellular free fattyacids result in increased cytosolic long chain CoA, the effects of whichinclude modulating protein kinase C (PKC) activity, intracellularprotein trafficking G-protein activity, endoplasmic reticulum (ER) Ca²⁺-ATPase activity, expression of acetyl-CoA carboxylase and peroxisomeproliferation (Prentki et al., supra; Prentki and Corkey (1996) supra;and Brun et al. (1996) 45:190-198).

Perhaps the most widely-accepted therapy for treating IDDM involvesdaily injection of insulin in combination with blood glucose monitoringand eating behavior modification, indirectly reducing undesirablesecondary side effects and the risk of life-threatening complications.Moreover, alternative therapies including pancreas and islettransplantation, autoantigen-based therapies (e.g., glutamic aciddecarboxylase (GAD) therapy), and β cell-related peptide adjunctivetherapies are being developed and tested. However, it is well-recognizedthat there remains a need for therapies that are more preventive innature, in particular, therapies aimed at correcting the underlyingabnormalities responsible for IDDM. Furthermore, there exists a need fornew diagnostic tools aimed at identifying persons having or who arepredisposed to the disease. In particular, there exists a need formethods of diagnosing persons at risk for developing IDDM, particularlyduring the long subclinical latency period associated with IDDM.

SUMMARY OF THE INVENTION

The present invention features novel methods of diagnosing persons orsubjects having diabetes or at risk for developing diabetes. Inparticular, the present invention features methods of diagnosingInsulin-Dependent Diabetes Mellitus (“IDDM”), also known as Type 1diabetes. The present invention is based, at least in part, on thediscovery of a striking difference in Ca²⁺ mobilization of human skinfibroblasts from patients with IDDM. In ten out of ten cultured celllines from unrelated subjects with IDDM, hyper-responsive Ca²⁺mobilization was observed as compared to the response in seven out ofseven unrelated control cell lines. Accordingly, in one embodiment thepresent invention features methods for diagnosing IDDM in a person orsubject (e.g., a test subject) which include detecting hyper-responsiveCa²⁺ mobilization in a cell sample obtained from the subject. In apreferred embodiment, detecting hyper-responsive Ca²⁺ mobilization incells obtained from the subject (e.g., the test subject) includescomparing Ca²⁺ mobilization in those cells to Ca²⁺ mobilization in cellsderived from a control subject. Ca²⁺ mobilization, according to thepresent invention is preferably induced by contacting cells with astimulatory agent (e.g., bradykinin).

Hyper-responsive Ca²⁺ mobilization was observed in cells particularly inresponse to treatments that are known to affect the expression of genesand proteins.

Exemplary treatments that caused hyper-responsive Ca²⁺ mobilization tobecome apparent were exposure to the inflammatory cytokines, TNF-α andIL-1β, that are elevated in newly diagnosed diabetics, and fatty acids,which are also elevated in diabetes. Accordingly, the methods of thepresent invention further feature contacting cells with a potentiatingagent in order to facilitate detection of hyper-responsive Ca²⁺mobilization in cells. In one embodiment, the potentiating agent is aninflammatory cytokine. Preferably, the potentiating agent is TNF-α orIL-1β. In another embodiment, the potentiating agent is a component ofthe diabetic milieu. Preferably, the potentiating agent is a free fattyacid (“FFA”), for example, oleate or oleic acid. It is also within thescope of the present invention to contact cells with at least twopotentiating agents, for example, prior to determining Ca²⁺mobilization. For example, cells (e.g., cells from a test subject and/orcells from a control subject) can be contacted or treated with aninflammatory cytokine (e.g., TNF-α or IL-1β) and a free fatty acid(e.g., oleic acid). These treatments change the Ca²⁺ signaling pathwaywhich plays a major role in cell growth and transmitting informationfrom the bloodstream to the interior of the cell.

Yet another aspect of the present invention includes methods foridentifying (e.g., diagnosing) subjects at risk for developing IDDM (orhaving IDDM, for example, subjects whose disease is in the preclinicallatency period) based on the striking difference in Ca²⁺ mobilization infibroblasts from these patients (e.g., as compared to control subjectsor to other standards). In one embodiment, the invention featuresidentifying a person or subject (e.g., a test subject) at risk ofdeveloping IDDM which includes comparing Ca²⁺ mobilization in cells(e.g., a test cell sample) obtained from the subject to, for example,Ca²⁺ mobilization in cells from a control subject. Preferably, theperson or subject at risk is identified by detecting a difference inCa²⁺ mobilization in the test cell sample as compared to the controlcell sample. In a preferred embodiment, a difference is detected in peakCa²⁺ response (e.g., to a stimulatory or inducing agent). In yet anotherpreferred embodiment, a difference is detected in steady state Ca²⁺following stimulation with an inducing or stimulatory agent. In a morepreferred embodiment, cells (e.g., test cells and/or control cells or analiquot thereof) are contacted with a potentiating agent (e.g., TNF-α orIL-1β), for example, prior to contacting with an inducing agent. In yetanother embodiment, a difference is detected in the Ca²⁺ responseincrement (e.g., the incremental increase in response between cellstreated with potentiating agent and untreated cells). Preferred subjectswhich benefit from the methodology described here are human subjects.The present invention further features kits for the diagnosis of IDDM orType 1 diabetes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph depicting bradykinin-induced Ca²⁺ mobilization infibroblasts isolated from control and diabetic human donors. Each barrepresents the mean of 14 to 33 separate determinations. Cross-hatchedbars represent cells isolated from control donors. Solid bars representcells isolated from diabetic donors.

FIG. 2 depicts the effect of exposure of cells to TNF-α (10 ng/ml for 24hours) on bradykinin-induced Ca²⁺ mobilization.

FIG. 2A depicts a trace from a control donor.

FIG. 2B depicts a trace from a diabetic donor. The traces depicted arerepresentative of traces obtained for all control and diabetic donors.

FIG. 3 depicts the effect of exposure of cells to IL-1β (1 ng/ml for 24hours) on bradykinin-induced Ca²⁺ mobilization.

FIG. 3A depicts a trace from a control donor.

FIG. 3B depicts a trace from a diabetic donor. The traces depicted arerepresentative of traces obtained for all control and diabetic donors.

FIG. 4 is a bar graph depicting the effect of exposure to TNF-α on peakresponse to bradykinin in control and diabetic human fibroblasts. Eachbar represents the mean of 14 to 33 separate determinations.Cross-hatched bars represent cells isolated from control donors. Solidbars represent cells isolated from diabetic donors. Diabetic issignificantly different from control (ANOVA p<0.001).

FIG. 5 is a bar graph showing the incremental effect of exposure toTNF-α on bradykinin-induced Ca²⁺ mobilization in human fibroblasts. Eachbar represents the mean of 14 to 34 separate determinations.Cross-hatched bars represent cells isolated from control donors. Solidbars represent cells isolated from diabetic donors. * indicates thatcontrol is significantly different from diabetic (ANOVA p<0.001).

FIG. 6 is a bar graph depicting the incremental effect of exposure toTNF-α on bradykinin-induced mobilization in human fibroblasts in 7control donors, 3 non-diabetic siblings, and 10 diabetic donors (3-8separate experiments for each donor). Each bar represents the mean of 6to 18 separate determinations.

FIG. 7 is a bar graph depicting the average incremental effect ofexposure to TNF-α on bradykinin-induced mobilization in humanfibroblasts. Each bar represents the mean of 18 to 52 separatedeterminations. Each groups is significantly different from others(ANOVA p<0.001).

FIG. 8 is a bar graph depicting the incremental effect of exposure toTNF-α on steady state Ca²⁺ following bradykinin stimulation humanfibroblasts. The magnitude of the final steady state, or restingintracellular Ca²⁺ following stimulation with bradykinin was determinedfor 7 control and 10 IDDM donors. Each bar represents the mean of 14 to34 separate determinations. Cross-hatched bars represent cells isolatedfrom control donors. Solid bars represent cells isolated from diabeticdonors. Diabetic cells are significantly different from control at theseconcentrations of bradykinin (ANOVA p<0.005).

FIG. 9 depicts the effect of EGTA on peak bradykinin response inuntreated and TNF-α treated fibroblasts. FIG. 9A depicts a trace from anuntreated cell population. FIG. 9B depicts a trace from a TNF-α treatedcell population. These are representative traces from one donor.

FIG. 10 depicts the effect of exposure to TNF-α on thapsigargin mediatedemptying of intracellular Ca²⁺ stores.

FIG. 10A depicts traces of thapsigargin-treated, fura-loaded fibroblastsfrom control donors.

FIG. 10B depicts traces of thapsigargin-treated, fura-loaded fibroblastsfrom diabetic donors.

FIG. 11 depicts a time course of the effect of TNF-α pre-treatment onpeak bradykinin responses. Fibroblasts from 3 different donors weretreated with TNF-α for 1 to 48 hr. After treatment, cells were loadedwith fura-2 AM and the TNF-α-induced increment in bradykinin responsedetermined. Each bar represents the mean of 2 to 6 separatedeterminations.

FIG. 12 depicts the effect of cyclohexamide on TNF-α-induced incrementin bradykinin response.

FIG. 13 depicts the effect of glucose and TNF-α on peak bradykininresponse in control fibroblasts. 11 mM glucose was added 24 hoursbefore, and throughout the subsequent 24 hour incubation. Each bar onthe graph represents the mean of 12 separate determination. TNF-α had asignificant effect on bradykinin response (ANOVA p<0.001), but 11 mMglucose did not.

FIG. 14 depicts the effect of glucose, oleate and TNF-α on peakbradykinin response in fibroblasts from an IDDM donor. These arerepresentative traces of bradykinin responses from one diabetic donor.Cells in 5.6 mM glucose were treated for 24 hr with TNF-α (10 ng/ml) orleft untreated. Where indicated, 11 mM glucose in combination with 2 mMoleate was added 24 hours before and throughout the subsequent 24 hourincubation. Following 24 hours of incubation, cells were loaded withfura-2 AM and response to 20 nM bradykinin was determined at 200seconds.

FIG. 15 depicts the effect of glucose, oleate, and TNF-α on peakbradykinin responses in human fibroblasts. Fibroblasts from 3 relativelyunresponsive control and 4 diabetic donors were treated for 24 hourswith TNF-α (10 ng/ml) or left untreated. Where indicated, 11 mM glucosein combination with 2 mM oleate was added 24 hours before, andthroughout the subsequence 24 hour incubation. Following 24 hours ofincubation, cells were loaded with fura-2 AM, and peak response tobradykinin was determined, expressed here as a percentage of theuntreated condition (5.6 mM glucose). Cross-hatched bars represent cellsisolated from control donors. Solid bars represent cells isolated fromdiabetic donors. Both TNF-α and oleate had significant effects on peakbradykinin response (ANOVA p<0.05 and p<0.001 respectively), anddiabetic fibroblasts responded differently from controls (p<0.001).

FIG. 16 depicts the effect of glucose, oleate, and TNF-α on steady state[Ca²⁺]i following bradykinin stimulation in human fibroblasts.Fibroblasts from 3 relatively unresponsive control and 4 diabetic donorswere treated with 24 hours of TNF-α (10 ng/ml) or left untreated. Whereindicated, 11 mM glucose in combination with 2 mM oleic acid was added24 hours before, and throughout the subsequent 24 hour incubation.Following 24 hours of incubation, cells were loaded with fura-2 AM, andsteady state [Ca²⁺]i following stimulation with bradykinin wasdetermined, expressed here as a percentage of the untreated condition(5.6 mM glucose). Cross-hatched bars represent cells isolated fromcontrol donors. Solid bars represent cells isolated from diabeticdonors. Oleic acid had a significant effect on steady state [Ca²⁺]i(p<0.001), and diabetic fibroblasts responded differently from controls(p<0.001).

DETAILED DESCRIPTION OF THE INVENTION

Highly significant differences in Ca²⁺ transients between cultured skinfibroblasts from ten unrelated Type 1 diabetic (IDDM) and seven controlsubjects were observed. The defect in Ca²⁺ signaling, referred to hereinas “hyper-responsive Ca²⁺ mobilization”, was found in cells from ten often patients with IDDM and none of seven controls. There was no overlapbetween the groups. Non-diabetic siblings exhibited intermediateresponses, suggesting a genetic basis for the effect. The three-foldenhancement was observed following exposure for a period of hours to acytokine such as TNFα or IL- 1 β or to free fatty acids (FFA) in thegrowth media. The changes were prevented by concurrent treatment withcyclohexamide, indicating the involvement of newly synthesized proteinsin the defect. Although bradykinin (BK) was used as an agonist tomonitor Ca²⁺ transients, the altered responses were not specific tobradykinin since the endoplasmic reticulum (ER) Ca²⁺ stores and the rateand extent of Ca²⁺ uptake were greater in the treated fibroblasts fromIDDM subjects than from controls.

Because the defect in Ca²⁺ mobilization (i.e., hyper-responsive Ca²⁺mobilization) is manifested in a common signal transduction pathway,mobilization of intracellular Ca²⁺, it is possible to identify novelspecific genes and/or proteins that are different between controls anddiabetics. Identification of these novel genes and/or proteins allowsthe development of screens to identify susceptible individuals and ofappropriate screens for testing potential therapeutic agents.Furthermore, the defect in the Ca²⁺ pathway is predicted to affectresponses to many hormones and agonists and because it is awell-understood pathway, provides a good target for testing theeffectiveness of putative drugs that might prevent the changes fromoccurring in susceptible individuals and potentially preventing thedevelopment of IDDM. Identification of these novel genes and/or proteinsallows the development of appropriate screens for testing potentialtherapeutic agents.

A. Diagnostic Assays

In one embodiment, the present invention involves a method fordiagnosing IDDM in a test subject which includes detectinghyper-responsive Ca²⁺ mobilization in cells obtained from the testsubject. The term “hyper-responsive Ca²⁺ mobilization” includes Ca²⁺responsiveness or Ca²⁺ mobilization in a cells, in particular,mobilization of Ca²⁺ from intracellular stores into the cytoplasm of acell. Ca²⁺ mobilization can result from contacting a cell, for example,with a stimulatory or inducing agent. The term “stimulatory agent” or“inducing agent” includes any compound or agent that causes a cell(e.g., induces, triggers, stimulates) a cell to mobilize Ca²⁺, forexample, from intracellular stores into the cytoplasm of a cell.“Stimulatory agents” or “inducing agents” of the present inventioninclude any agent that acts on a cell through PLC activation. Morepreferably, inducing agents include any agent that causes release ofcalcium from the ER of a cell. Exemplary preferred inducing agentsinclude bradykinin, epinephrine, calcium ionophores and the like. Alsopreferred are, for example, agents that inhibit Ca²⁺-ATPase responsiblefor sequestering calcium in the ER (e.g., thapsigargin). Cells usefulaccording to the diagnostic methods of the present invention include anycell obtained or isolated form the subject (e.g., test subject orcontrol subject) which is capable of mobilizing calcium. Preferred cells(e.g., test cells and/or control cells include fibroblasts, for examplefibroblasts cultured from a skin biopsy, white blood cells, for exampleperipheral blood leukocytes, fat cells, and the like. Cells can exist inpopulations as cultures or as single cells (e.g., with Ca²⁺ mobilizationdetected via an imaging system).

The phrase “detecting hyper-responsive Ca²⁺ mobilization” includesdetecting any indicator of the trait defined herein as hyper-responsiveCa²⁺ mobilization. For example, Ca²⁺ mobilization can be determined in acell according to any methodology familiar to one of ordinary skill inthe art and compared to any suitable control or standard to determinethat Ca²⁺ mobilization (e.g., Ca²⁺ mobilization in the test cell) ishyper-responsive. Ca²⁺ mobilization can be determined, for example, byloading cells with a calcium-sensitive dye (e.g., a fluorescent orcolorimetric calcium-sensitive dye). A preferred calcium-sensitive dyeis fura-2 acetoxymethyl ester, also referred to herein as fura-2 AM orfura-2. An exemplary Ca²⁺ mobilization response or Ca²⁺ responseincludes an increase and peak in intracellular Ca²⁺ concentrationfollowed by a decrease and plateau in intracellular calciumconcentration. The initial intracellular Ca²⁺ concentration is referredto herein and in the art as the basal intracellular Ca²⁺ concentration.The ending intracellular Ca²⁺ concentration is referred to as steadystate intracellular Ca²⁺ concentration. As described previously,“detecting hyper-responsive Ca²⁺ mobilization” can include determiningCa²⁺ mobilization in a cell according to any methodology familiar to oneof ordinary skill in the art and comparing it to any suitable control orstandard to determine that Ca²⁺ mobilization is hyper-responsive. In apreferred embodiment, “detecting hyper-responsive Ca²⁺ mobilization”involves comparing Ca²⁺ mobilization in cells obtained from the testsubject to Ca²⁺ mobilization in cells obtained from a control subject.Accordingly, the suitable control can be a control cell. The phrase“control cell” includes any cell which exhibits normal traits, ascompared to diabetic traits (e.g, hyper-responsive Ca²⁺ mobilization).In one embodiment, control cells are cells of the same cell type as thetest cell (or cell obtained or isolated from the test subject) but areobtained or isolated from a control subject (e.g., a subject devoid ofIDDM or traits thereof). In another embodiment, control cells are cellsobtained or isolated from the test subject but which exhibit normaltraits. It is also within the scope of the present invention to usecontrol cells, for example cell lines or cultures, which have predefinedcharacteristics (e.g., have been previously determined to exhibit anormal phenotype). As defined herein, a suitable control can alsoinclude a predefined indication of normal phenotype. For example, anormal intracellular Ca²⁺ concentration, for example, for a particularcell type, can be predetermined form analysis of normal cells and thatindication used as a control according to the present methodology. Inone embodiment, a normal peak intracellular Ca²⁺ concentration can bedetermined (e.g., following bradykinin stimulation of normal cells) andthat number (taking into account reasonable variation) can be used as asuitable control.

The term “detecting hyper-responsive Ca² ⁺ mobilization” furtherincludes detecting any protein characteristic of the hyper-responsiveCa²⁺ mobilization trait. For example, the present inventors havedemonstrated that hyper-responsive Ca²⁺ mobilization is inhibitable byprotein synthesis inhibitors. Accordingly, one of ordinary skill in theart can characterize proteins involved in conferring thehyper-responsive Ca²⁺ mobilization trait on cells and detect theabundance or activity of such proteins as indicating of thehyper-responsive Ca²⁺ mobilization trait.

In another aspect, the method includes the step of contacting the cellswith a potentiating agent prior to comparing Ca²⁺ mobilization.Preferred potentiating agent include inflammatory cytokine (e.g., TNF-α,IL-1β, IFN-γ or LIF) as well as certain components of the diabeticmilieu (e.g., free fatty acid (FFA), for example, oleic acid). Thephrase “diabetic milieu” includes the extracellular environmentexperienced by a cell, for example, a cell within a diabetic donor.Components of the “diabetic milieu” include any agent that elevatescytosolic free calcium from intracellular stores and preferably caninclude free fatty acids and/or high glucose. Preferred components ofthe diabetic milieu include, for example, oleic acid.

Preferred methods of the present invention can further includecontacting the cells with two potentiating agents prior to comparingCa²⁺ mobilization (e.g., TNF-α or IL,-1 β and FFA). The use of apotentiating agent is particularly desirable due to the fact thatdifferences in Ca²⁺ mobilization between normal and control cells areaugmented by inclusion of the potentiating agent. For example, peak Ca²⁺mobilization response in cells from diabetic donors differs from that ofcontrol cells (e.g., is enhanced) when both are treated with apotentiating agent. Moreover, steady state Ca²⁺ concentrations inbradykinin-induced cells from diabetic donors differ from that ofcontrol cells when both are treated with a potentiating agent.Furthermore, Ca²⁺ mobilization increments (e.g., the incrementalincrease detected when comparing cells in the presence versus absence ofpotentiating agent) are detectable only when cells (or at least analiquot of cells) are treated with potentiating agent. As exemplifiedherein, potentiation is a time-dependent effect. Accordingly, when cellsare treated with a potentiating agent, they are preferably treated forat least 1 hour, preferably 2 hours, more preferably 3-4 hours, evenmore preferably between 4 and 12 hours, even more preferably between 12and 24 hours or greater than 24 hours.

In another embodiment, the present invention involves a method foridentifying a subject at risk of developing IDDM or a subject havingIDDM which includes obtaining a test cell sample from a test subject,determining Ca²⁺ mobilization in the test cell sample, comparing theCa²⁺ mobilization in the test cell sample to Ca²⁺ mobilization in acontrol cell sample from a normal subject, and identifying a subject atrisk of developing IDDM or a subject having IDDM by detecting adifference in Ca²⁺ mobilization in the test cell sample as compared tothe control cell sample. In a preferred embodiment, the test cell sampleand the control cell sample are contacted with a stimulatory agent(e.g., bradykinin) to induce Ca²⁺ mobilization. In another embodiment,the test cell sample and the control cell sample are contacted with apotentiating agent prior to stimulation with the stimulatory agent. Apreferred potentiating agent is, for example, an inflammatory cytokine(e.g., TNF-α or IL-1β).

In another embodiment, the present invention involves a method foridentifying a subject at risk of developing IDDM or a subject havingIDDM which includes obtaining a test cell sample from a test subject,contacting the test cell sample with a stimulatory agent (e.g.,bradykinin), determining steady state Ca²⁺ levels in the test cellsample following response of the cell to the stimulatory agent,comparing the steady state Ca²⁺ levels in the test cell sample followingresponse of the cell to the stimulatory agent to steady state Ca²⁺levels in a control cell sample from a normal subject following responseof the control cell to the stimulatory agent, and identifying a subjectat risk of developing IDDM or a subject having IDDM by detecting adifference in steady state Ca²⁺ levels in the test cell sample ascompared to the control cell sample. In another embodiment, the testcell sample and the control cell sample are contacted with apotentiating agent prior to stimulation with the stimulatory agent. Apreferred potentiating agent is, for example, an inflammatory cytokine(e.g., TNF-α or IL-1β).

In yet another embodiment, the present invention involves a method foridentifying a subject at risk of developing IDDM or a subject havingIDDM which includes obtaining a test cell sample from a test subject,contacting the test cell sample with at least one component of thediabetic millieu (e.g., FFA and/or glucose), determining Ca²⁺mobilization in the test cell sample, comparing the Ca²⁺ mobilization inthe test cell sample to Ca²⁺ mobilization in a control cell sample froma normal subject following response of the control cell to at least onecomponent of the diabetic millieu, and identifying a subject at risk ofdeveloping IDDM or a subject having IDDM by detecting a difference inCa²⁺ mobilization in the test cell sample as compared to the controlcell sample. In a preferred embodiment, the test cell sample and thecontrol cell sample are contacted with a stimulatory agent (e.g.,bradykinin) to induce Ca²⁺ mobilization. In another embodiment, the testcell sample and the control cell sample are contacted with apotentiating agent prior to stimulation with the stimulatory agent. Apreferred potentiating agent is, for example, an inflammatory cytokine(e.g., TNF-α or IL-1β).

In yet another embodiment, the present invention involves a method foridentifying a subject at risk of developing IDDM or a subject havingIDDM which includes obtaining a test cell sample from a test subject,contacting the test cell sample with at least one component of thediabetic millieu (e.g., FFA and/or glucose), contacting the test cellsample with a stimulatory agent (e.g., bradykinin), determining steadystate Ca²⁺ levels in the test cell sample, comparing steady state Ca²⁺levels in the test cell sample to steady state Ca²⁺ levels in a controlcell sample from a normal subject following response of the control cellto at least one component of the diabetic millieu and the stimulatoryagent, and identifying a subject at risk of developing IDDM or a subjecthaving IDDM by detecting a difference in steady state Ca²⁺ levels in thetest cell sample as compared to the control cell sample. In anotherembodiment, the test cell sample and the control cell sample arecontacted with a potentiating agent prior to stimulation with thestimulatory agent. A preferred potentiating agent is, for example, aninflammatory cytokine (e.g., TNF-α or IL-1β).

Examplary methods of determining mobilization of Ca²⁺ and steady stateCa²⁺ levels in cells are described in detail in the following examples.Furthermore, it is intended that such determinations can be made usingalternative methods known in the art for determining mobilization ofCa²⁺ and steady state Ca²⁺ levels in cells.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all referencescited throughout this application are hereby incorporated by reference.

EXAMPLES

The following examples describe novel findings upon which the presentinvention is based, at least in part. In particular, the presentinventors have determined the effects of tumor necrosis factor alpha(TNF-α) treatment, and the diabetic environment (elevated glucose andfatty acid), on bradykinin-induced Ca²⁺ mobilization in dermalfibroblasts from type 1 diabetic patients and matched controls.Fibroblasts were exposed to TNF-α (10 ng/ml) for up to 48 hours. Cellsin suspension were then loaded with fura-2 acetoxymethyl ester, andbradykinin-induced Ca²⁺ mobilization was measured using fluorescencespectrophotometry. Basal intracellular Ca²⁺ levels were significantlylower in diabetic fibroblasts than controls (P<0.05), and TNF-αtreatment caused a significant increase in basal Ca²⁺ in diabetic butnot control cells (p<0.05). Beginning with 1 hour of TNF-α treatment,increases in Ca²⁺ mobilization in response to bradykinin (1 nM to 1 μM)were observed in cells from both controls and diabetics. With 24 hoursof treatment, TNF-α-induced increments in peak bradykinin response werethree-fold greater in diabetics than in controls (P<0.001). Similarresults were seen with interleukin-1 beta (IL-1β) treatment. Ca²⁺transients induced by thapsigargin, an inhibitor of the endoplasmicreticulum Ca²⁺-ATPase, were also greater in TNF-α treated fibroblaststhan in untreated cells, with an apparently greater increase in cellsfrom diabetic donors. These data indicate that TNF-α caused an increasein intracellular Ca²⁺ stores, which affected the magnitude ofagonist-induced Ca²⁺ responses. Exposing fibroblasts to a combination of11 mM glucose and 2 mM oleic acid for 48 hours caused increases in boththe peak bradykinin response and the TNF-α induced increment in peakresponse, which were significantly greater in diabetics than controls(p<0.001); 11 mM glucose alone was without effect. That these phenomenawere exhibited to a higher degree in cells from type 1 diabetics than incontrol cells indicates that fibroblasts from diabetic patients have aheightened sensitivity to TNF-α and oleic acid.

Example 1 Cells From Diabetic Donors Exhibit Hyper-Responsive CalciumMobilization as Compared to Cells From Control Donors

As an exemplary agonist of calcium mobilization, bradykinin was firstdemonstrated to induce calcium mobilization in cells isolated from bothcontrol and diabetic donors. Briefly, fibroblasts from 7 control and 10diabetic donors (passages 7 to 30) were loaded with fura-2 AM and Ca²⁺mobilization in response to increasing concentrations of bradykinin wasdetermined. As shown in FIG. 1, stimulation of cells from both controland diabetic with 200 nM bradykinin resulted in maximal Ca²⁺mobilization (peak response), with detectable Ca²⁺ mobilizationresulting from 20 nM bradykinin stimulation.

Inflammatory cytokines such as TNF-α and IL-1β have previously beenshown to modulate bradykinin responsiveness in varied experimentalsystems (O'Neill, Lewis (1989) Eur. J. Pharmacol. 166:131-137 and Amraniet al. (1995) Brit. J. Pharmacol. 144:4-5). In fibroblasts isolated fromcontrol and diabetic donors, a 24 hour incubation with TNF-α (10 ng/ml)potentiated bradykinin-induced Ca²⁺ mobilization, as shown in FIG. 2.Briefly, paired flasks of cells were treated with TNF-α or leftuntreated. Fura-loaded cells were stimulated with 20 nM bradykinin andCa²⁺ mobilization was measured after 200 seconds. Similar results wereseen with IL-1β treatment (1 ng/ml for 24 hours) (FIG. 3). FIG. 4 showsthe effect of TNF-α potentiation (10 ng/ml for 24 hours) on the peakbradykinin-induced Ca²⁺ mobilization response in fibroblasts isolatedform 7 control and 10 diabetic (passages 7 to 30) donors. Fibroblastswere fura-2 AM loaded and Ca²⁺ mobilization in response to increasingconcentrations of bradykinin was determined. Each bar represents themean of 14 to 33 separate determinations. In cells from both control anddiabetic donors, TNF-α treatment augmented bradykinin induced Ca²⁺mobilization, although to a much greater extent in the diabetic donor.Statistical analysis of the data indicate that the Ca²⁺ mobilizationresponse in diabetics is significantly different from controls (ANOVAp<0.001). A small increase in the final equilibrium level of Ca²⁺ wasalso seen consistently in the diabetic fibroblasts, but not in thecontrols.

The difference between control and diabetic cells can be seen moreclearly through the bradykinin-induced increment between untreated andTNF-α treated.

FIG. 5 shows the incremental effect of exposure to TNF-α (10 ng/ml for24 hours) on bradykinin-induced Ca²⁺ mobilization in fibroblastsisolated from control versus diabetic humans. The change in peakresponse to bradykinin after TNF-α treatment was measures in fibroblastsfrom 7 control and 10 diabetic donors (3-8 separate experiments perdonor). Each bar on the graph represents the mean of 14 to 34 separatedeterminations. When the difference between TNF-α treated and untreatedincrements was compared, the diabetic cells showed a 3-fold greatereffect of bradykinin-induced Ca²⁺ mobilization than control cells. ANOVAindicated an overall difference between control and diabetic (p<0.001),and specific differences between control and diabetic (Tukey p<0.001) atevery bradykinin concentration above 1 nM.

FIG. 6 shows this increment in all of the donors examined. The incrementin peak response to bradykinin following TNF-α treatment was determinedin 7 control donors, 3 non-diabetic siblings of type 1 diabetics and 10diabetic donors (3-8 separate experiments per donor). Each barrepresents the mean of 6 to 18 separate determinations. The average ofthese data are shown in FIG. 7. The increments in peak response werepooled (3-8 experiments for each donor). Each bar represents the mean of18 to 52 separate determinations. Each group is significantly differentfrom the others (ANOVA p<0.001).

Examination of non-diabetic siblings. As the theories of thepathogenesis of IDDM indicated there is some genetic component to thedisease, 3 non-diabetic siblings of the 10 previously described diabeticdonors (from different families) were obtained to determine whether thenon-diabetic siblings more closely resembled their diabetic familymembers, or their control counterparts in terms of the effects of TNF-αon bradykinin-induced Ca²⁺ mobilization. These subjects are shown in themiddle 3 bars of FIG. 6 with average values presented in FIG. 7 of the 7original control donors, the 10 original diabetic donors and 3non-diabetic siblings of the diabetics (passages 8 to 15). As can beseen, the non-diabetic siblings of diabetics have a response to TNF-αthat is intermediate between the two other groups.

A brief analysis of the TNF-α induced increment in peak bradykininresponse in all the donors surveyed showed that the donors can be rankedinto 3 different groups, based on the magnitude of their bradykininresponse following a TNF-α treatment. Donors with a TNF-α inducedincrement of less than 200 nM Ca²⁺ can be described as low responders,donors in which the TNF-α induced increment was between 200 and 300 nMCa²⁺ can be characterized as intermediate responders, and those donorsin which the TNF-α induced increment was greater than 300 can bedescribed as high responders. Most the control donors fall into the lowresponder category, while most of the diabetic donors fall into the highresponder category; all of the non-diabetic siblings, and a lowpercentage of both the controls and the diabetics fall into theintermediate responder category.

This artificial separation into three ordinal groups, according toresponse to treatment can illuminate possibilities as to the causationof FIDDM, and the lack of a strong concordance rate among siblings for asuspected genetic disease. All of the diabetic donors, and theirsiblings fall into either the medium or high responders categories,there are no low responders. IDDM comprise a very small percentage ofthe general population, probably not greater than 1% (Cotran et al.(1989) Ribbins Pathologic Basis of Disease 994-1005), and 10 randomlyselected fibroblast donors all exhibit greater effects of TNF-α thanmatched control donors. A trait present in 100% of such a smallpopulation must also occur with some frequency in the generalpopulation: this explains why some of the control donors approach, orenter the intermediate responder classification. If the causation ofdiabetes does in fact require a combination of different factors, thiscan explain why two siblings who both carry a “diabetes gene” can bediscordant for the disease. For example, if a genetic predisposition toTNF-α hypersensitivity (which seems to be the case in the diabeticdonors, and their siblings studies here, in terms of the TNF-α-inducedincrement in bradykinin response) mediates autoimmune destruction ofpancreatic β cells, then both siblings carrying the hypersensitivitytrait would have to encounter the appropriate trigger (e.g., a specificsystemic viral infection) to raise TNF-α in the pancreas high enough tomediate insularities. If one sibling contracted a less virulentinfection, or avoided the infection all together, insulitis would notoccur at that time. A person who does not carry the hypersensitivitytrait would not become diabetic by this mechanism, whether or not theappropriate systemic viral injection occurred.

The presence of low, intermediate, and high response groups resemblesthe effects of autosomal recessive genetic traits. If TNF-αhypersensitivity reflects an autosomal recessive gene, then one wouldhave to be homozygously recessive to be in the high responder group.Homozygous dominant individuals would be low responders, and personsheterozygous for the trait could exhibit incomplete dominance, and beintermediate responders. In this way, a control donor, with no familyhistory of diabetes, could still carry the recessive trait, and be anintermediate responder to TNF-α. Diabetic donors could be intermediateor high responders based on whether they were heterozygous, orhomozygous for the trait. If an individual was homozygous recessive, anda hyper-responder to TNF-α, there would be less chance of escapingchildhood, and the many viral injections encountered therein, withoutdeveloping insulities and diabetes. A heterozygous, intermediateresponder, however, may encounter the same injections and not developinsulities, because their cells would not respond as vigorously to thesame amount of TNF-α.

Example 2 Hyper-Responsive Calcium Mobilization can be Detected as anIncrease in Steady State Ca²⁺ in TNF-α-Treated Fibroblasts FollowingStimulation with Bradykinin

Following the initial bradykinin induced peak of Ca²⁺ mobilized from theER, a sustained elevation of Ca²⁺ occurs lasting for minutes. Thissustained elevation in steady state Ca²⁺ requires Ca²⁺ entry from theextracellular space. Inappropriate elevation of Ca²⁺ may lead to cellinjury, apoptosis and death by activation of phospholipases, nucleases,and proteases (Cheung et al. (1986) N. Engl. J Med. 314:1670-1676):thus, inappropriately elevated Ca²⁺ in diabetic cells could be animportant mechanisms for initiating diabetic pathology. Because of theimportant signal transducing properties of the sustained phase of theCa²⁺ response, the effects of TNF-α treatment on the steady state wereexamined. Briefly, fibroblasts from 7 control and 10 diabetic donorswere exposed to 10 ng/ml TNF-α for 24 hours prior to bradykininstimulation. The magnitude of the final steady state, or resting,intracellular [Ca²⁺] following stimulation with bradykinin wasdetermined. The incremental effect of exposure to TNF-α was examined andthe results are shown in FIG. 8. Each bar represents the mean of 14 to34 separate determinations. TNF-α treatment caused significantelevations of steady state Ca²⁺ following the bradykinin response inboth control and diabetic donors (ANOVA p<0.001) which weresignificantly higher in the diabetic fibroblasts than in the controls(ANOVA p<0.005).

Example 3 Effect of EGTA on TNF-α-Induced Increases in Peak BradykininResponse

To determine whether the TNF-α induced increase in the peak bradykininresponse was due to mobilization of Ca²⁺ from intracellular stores, theCa²⁺ chelator EGTA was added prior to stimulation with bradykinin. FIG.9 illustrates an experiment in which fibroblasts were treated with TNF-αfor 24 hours or left untreated and the peak bradykinin response measuredin the presence or absence of 2 mM EGTA. In these traces, 2 mM EGTA wasadded 10 seconds prior to stimulation with 200 nM bradykinin. The peakresponse was unaltered by EGTA in untreated and TNF-α treated cells.(EGTA did, however, eliminate the increase in final [Ca²⁺]I). Thisindicated that extracellular Ca²⁺ did not contribute to the bradykinininduced Ca²⁺ peak, and was not responsible for the TNF-α inducedincrement in peak response.

Example 4 Effect of TNF-α on Thapsigargin Emptying of Intracellular Ca²⁺Stores

Thapsigargin, an irreversible inhibitor of the intracellularCa²⁺-ATPase, was used to determine if the TNF-α induced increment inpeak bradykinin response was mediated by changes in the bradykininsignal transduction cascade, or was at the level of the intracellularCa²⁺ stores by irreversibly inhibiting their refilling. FIG. 10illustrates representative traces in which thapsigargin was added tountreated and TNF-α treated cells from control and IDDM donors. Additionof thapsigargin caused Ca²⁺ to leak out of the ER, resulting in atransient rise of cytosolic Ca²⁺, which plateaued at a new set point. Inboth control and IDDM fibroblasts treated with TNF-α treatment had agreater effect in the diabetic donors than in the controls. Potentialmechanisms include alterations in modulation of Ca²⁺-ATPase activity,changes in the ATPase itself or alterations in intraluminal signaling.

Example 5 Time is Required for TNF-α Treatment to AffectBradykinin-Induced Ca²⁺ Mobilization

TNF-α treatments did not have an immediate effect on Ca²⁺ mobilization,but within 24 hours of treatment, a clear and dramatic effect was seen.To determine the time required for TNF-α to induce an effect on peakbradykinin response, a series of time courses were done in 3 differentdonors (passages 14 to 27), in which fibroblasts were treated with 10ng/ml TNF-α for 1, 2, 4, 12, 24 or 48 hours. FIG. 11 shows the resultsof these experiments. Each bar on the graph represents the mean ofbetween 2 and 6 separate determinations. In these donors, a TNF-αinduced increment in peak bradykinin response could be seen within a fewhours of treatment. A maximum increment was achieved by 24 hour that didnot diminished by 48 hr.

These data indicate that time is required for TNF-α to cause anincrement in peak bradykinin response, consistent with the possibilityof increased protein expression. Consistent with this notion, theprotein synthesis inhibitor cycloheximide blocked the TNF-α inducedincrement (FIG. 12). Changes in calreticulin expression can modulate theresponses of Ca²⁺ mobilizing agonists (Liu et al. (1994) J Biol. Chem.269:28635-28639), however, an increase in calreticulin expression didnot occur; Western blots shown no change in calreticulin expression didnot occur; Western blots shows no change in calreticulin expressionafter 24 hr of TNF-α treatment or between control and diabeticfibroblasts (data not shown). Although the cycloheximide data indicatesthat synthesis of new proteins is required for the TNF-α effect, itcannot be concluded from this that TNF-α is responsible for inducinggene expression to achieve that effect. Although TNF-α is known to turnon a multitude of genes in many different cell types, including thefibroblast (Tessier et al. (1993) Arthritis & Rheumatism 36:1528-1539;Rathanaswami et al. (1993) Arthritis & Rheumatism 36:1295-1304; Butleret al. (1994) Eur. Cytokine Network 5:441-448), it is possible that newprotein synthesis is required to replete a component of the signaltransduction apparatus that is constantly being turned over.

Example 6 Effect of the Diabetic Milieu on TNF-α Induced Peak BradykininResponses

Tight metabolic control of diabetes is recommended to prevent the onsetof diabetic pathologies. There is much evidence that suggests that thediabetic environment, specifically, elevated serum glucose levels, isresponsible for the generation of these pathologies. The goal of thisset of experiments was to assess the effect of a simulated diabeticenvironment. First, the effects of elevated glucose levels (11 mM) wereexamined in control and diabetic fibroblasts by comparison to the effectof TNF-α on bradykinin responses in the normal experimental medium (5.6mM glucose). Fibroblasts were exposed to the experimental mediumcontaining 11 mM glucose for 48 hours. TNF-α treated groups were exposedto TNF-α for the final 24 hours of 48 hour treatment period. Four groupswere compared for each donor examined: 5.6 mM glucose alone, 5.6 mMglucose plus TNF-α, 11 mM glucose alone, and 11 mM glucose plus TNF-α.The results of these experiments are shown in FIG. 13. Elevated glucosedid not affect controls and diabetics differently, so the results from 3control donors and 3 diabetic donors (passages 13 to 31) were pooled inthis figure. Each bar represents the mean of 12 separate determinations.TNF-α did have its usual significant elevation of peak bradykininresponse (p<0.001), but glucose was without further significant effect.

Another component of the diabetic milieu is elevated free fatty acid(FFA) in the serum. To examine the effects of FFA on bradykinin inducedCa²⁺ mobilization and on the TNF-α induced increment, fibroblasts from 4diabetic and 3 control donors (passages 12 to 23) were treated with 2 mMFFA plus 11 mM glucose (from hereon termed FFA medium) for 48 hours.TNF-α treated groups were exposed to TNF-α for the final 24 hours of the48 hour treatment period. Four groups were compared for each donorexamined: 5.6 mM glucose alone, 5.6 mM glucose plus TNF-α. FIG. 14 showsrepresentative traces from an experiment performed on fibroblasts from asingle diabetic donor. As can be seen from the superimposed traces(representing the 4 difference treatment conditions), each of thetreatments had an effect on both peak bradykinin response, and thesustained phase of the response. Traces from parallel experiments incontrol donors showed little or no effect of TNF-α and a small effect ofthe FFA medium, but with less than 25% difference between the lowest(untreated) and highest (FFA medium +TNF-α) peaks (data not shown). Theresults of the experiments performed in 3 control and 4 diabetic donorswere pooled, and are shown in FIG. 15. Each bar on the graph representsthe mean of 6 or 8 separate determinations. Peak responses to bradykininwere normalized to a % of 5.6 mM alone.

In experiments to test the effects of the diabetic milieu, the controldonors chosen were picked because they had responded to TNF-α treatmentwith only modest increases in peak bradykinin response in previousexperiments and, thus, could exhibit a greater increment in response totreatment. In addition, the 2% BSA used in the media could potentiallybinding a portion of the TNF-α, possibly explaining the failure of thecontrol fibroblasts to respond vigorously to the TNF-α treatment. PairedT-tests indicated that the effects of the combination of diabeticenvironment and TNF-α were greater than those of diabetic environmentalong (p<0.01), and that there was no significant difference between theeffects of TNF-α and the diabetic environment. The response of diabeticcells in this medium were significantly greater than control cells.These test media contained both high glucose levels. If control cellshad responded like diabetic cells, inferences could have been made tosuggestive of a diabetic environment playing a major role in theetiology of IDDM. As high glucose alone did not impact the bradykininresponse, the effects of the diabetic medium were most likely due to thepresence of FFA.

The major findings in this work can be summarized as follows: 1)fibroblasts from diabetic donors had lower resting Ca²⁺ than fibroblastsfrom control donors; 2) TNF-α treatment (10 ng/ml for 24 hours) inducedan increment in peak bradykinin response (due to increased size of theintracellular stores) and in steady state Ca²⁺ following stimulationwith bradykinin, that were significantly greater in diabetic cells thanin control cells; 3) the high glucose/high fatty acid environment causedan increment in both peak bradykinin response and steady state Ca²⁺following stimulation with bradykinin, and diabetics respondeddifferently from controls in this environment. These findings areincorporated into the following model: IDDM fibroblasts have adeficiency in lipid metabolism which leads to the buildup of long chainacyl CoA, which in turn activates the ER Ca²⁺-ATPase, lowering basalCa²⁺. TNF-α and IL-1β inhibit oxidation of fatty acids (Corkey et al.(1988) J. Clin. Invest. 82:782-788; Kilpatrick et al. (1989) Metabolism38:73-77; Nachiappan et al. (1994) Shock 1:123-129), leading to afurther buildup of cytosolic long chain acyl CoA, further increasingCa²⁺-ATPase activity, and increasing the size of the bradykininmobilizable Ca²⁺stores (Deeney et al. (1992) J. Biol. Chem.267:19840-19845), and bradykinin response by the same mechanism. Asdiabetic fibroblasts have this preexisting deficiency in lipidmetabolism, the effects of TNF-α and oleic acid on bradykininmobilizable Ca²⁺ stores are exaggerated, as compared to controlfibroblasts.

We claim:
 1. A method for diagnosing IDDM in a test subject comprisingdetecting hyper-responsive Ca²⁺ mobilization in cells obtained from thetest subject as compared to a suitable control, wherein detection ofhyper-responsive Ca²⁺ mobilization correlates with the diagnosis ofIDDM.
 2. The method of claim 1, wherein detecting hyper-responsive Ca²⁺mobilization comprises comparing Ca²⁺ mobilization in cells obtainedfrom the test subject to Ca²⁺ mobilization in cells obtained from acontrol subject.
 3. The method of claim 2, wherein comparing Ca²⁺mobilization comprises contacting the cells with a stimulatory agent toinduce Ca²⁺ mobilization.
 4. The method of claim 3, wherein thestimulatory agent is bradykinin.
 5. The method of claim 2, furthercomprising contacting the cells with a potentiating agent prior tocomparing Ca²⁺ mobilization.
 6. The method of claim 5, wherein thepotentiating agent is an inflammatory cytokine.
 7. The method of claim6, wherein the potentiating agent in TNF-α.
 8. The method of claim 6,wherein the potentiating agent is IL-1β.
 9. The method of claim 5,wherein the potentiating agent is a component of the diabetic milieu.10. The method of claim 9, wherein the potentiating agent is FFA. 11.The method of claim 10, wherein the potentiating agent is oleate. 12.The method of claim 11, comprising contacting the cells with twopotentiating agents prior to comparing Ca²⁺ mobilization.
 13. A methodof identifying a subject at risk of developing IDDM comprising comparingCa²⁺ mobilization in a test cell sample obtained from the subject toCa²⁺ mobilization in a control cell sample and identifying the subjectat risk by detecting a difference in Ca²⁺ mobilization in the test cellsample as compared to the control cell sample.
 14. The method of claim13, wherein detecting a difference in Ca²⁺ mobilization comprisescomparing peak Ca²⁺ response in the test cell sample to peak Ca²⁺response in the control cell sample.
 15. The method of claim 13, whereindetecting a difference in Ca²⁺ mobilization comprises comparing steadystate Ca²⁺ in the test cell sample to steady state Ca²⁺ in the controlcell sample.
 16. The method of claim 13, wherein the test cell sampleand the control cell sample are contacted with an inducing agent toinduce Ca²⁺ mobilization.
 17. The method of claim 13, further comprisingcontacting the test cell sample with a potentiating agent.
 18. Themethod of claim 13, wherein comparing Ca²⁺ mobilization in the test cellsample to Ca²⁺ mobilization in the control cell sample comprisescomparing an incremental increase in Ca²⁺ mobilization in the test cellsample in the presence and absence of potentiating agent to anincremental increase in Ca²⁺ mobilization in the test cell sample in thepresence and absence of potentiating agent.
 19. The method of claim 1 or13 wherein the test subject is a human subject.
 20. A kit for thediagnosis IDDM in a subject or the risk thereof, comprising a controlcell sample, a stimulatory agent, a potentiating agent and instructionsfor use.